Efficient regulation of translation is a hallmark of human cells as it affords precise control over rates of cellular growth and proliferation. Cancer cells commonly display a dysregulation of translational control, often through increased expression of ribosomal proteins and rRNAs or increased signaling through pro-growth and proliferation pathways. One of these signaling cascades, the PI3K-mTOR pathway, regulates protein synthesis in part through its activation of the initiation factor eIF4A, an RNA helicase which increases rates of translation initiation through the local unwinding of secondary structure within the 5'-untranslated region (5'-UTR) of messenger RNAs and allows for their translation at the ribosome. Recent studies have identified multiple other RNA helicases that play non-redundant roles in mRNA translation, but the substrate specificity of these enzymes, as well as their roles in tumorigenesis, is not well understood. This proposal aims to develop novel chemical tools for the study of RNA helicases and will use these tools to uncover the scope of regulation of translation by RNA helicases. We hypothesize that genes with common functions contain conserved secondary structural elements within the 5'-UTR of their mRNA and that this structured RNA is recognized by a specific RNA helicase, which in turn is critical for regulating the translation of this gene subset. Aim 1 will develop an electrophile-sensitive chemical genetic approach based on covalent complementarity to specifically inhibit a single RNA helicase in a cellular environment with a small molecule. Preliminary studies suggest that a cysteine can be mutated into the ATP-binding site of RNA helicases and that this electrophile- sensitive (ES) RNA helicase is uniquely sensitive to NMS-859-based small molecule electrophiles. We will first test the generalizability of this cysteine-mutation across all subsetsof RNA helicases to ensure that they are active biochemically and in cells. Concurrently, we will synthesize derivatives of NMS-859 to increase potency for ES RNA helicases while reducing off-target reactivity. Aim 2 will assess the role of individual RNA helicases in translational control. Electrophile-sensitive versions of the RNA helicases implicated in translational control (eIF4A, DDX3X, DDX4, DDX6, DHX29, DDX43) will be individually introduced into otherwise isogenic cell lines with CRISPR-mediated genome engineering. Ribosomal profiling in the presence of ES RNA helicase inhibitors will be used to uncover the scope of regulation of translation by each individual RNA helicase. Completion of this proposal will elucidate the regulation of translation by mRNA secondary structure and RNA helicases, and provide a set of novel therapeutic targets to limit the dysregulation of translation in cancer.